The goal of this program is to obtain a better understanding of the mechanisms of insulin degradation and of the role of degradation in the biological action of insulin. In regard to mechanisms the hypothesis is that proteolytic processes are important in insulin degradation and that the enzyme insulin protease is the primary proteolytic enzyme. Additional investigation and characterization of this enzyme therefore is necessary. In addition examination of the mechanisms of insulin degradation will include identification of the products of degradation and the sequence and kinetics of the appearance of the products. The initial cleavage of insulin by this enzyme is in the B chain between B16-B17. This results in a molecule with the same molecular weight as insulin but consisting of three peptide chains held together by disulfide bonds. The properties of this molecule, including potential biological activity, have not been determined. We will prepare this material, purify it, and examine in detail its properties. In addition to studies on the initial product of insulin protease, subsequent cleavages will be identified and the properties of these products examined. These products will also be characterized as to their biological activity in order to examine the hypothesis that degradation might be involved in insulin action by producing an active fragment. Further examination of this hypothesis and the alternative hypothesis - that degradation is involved in the termination of insulin action - will be by studying insulin metabolism in intact cells and tissues. Relationships of degradation to insulin binding and action will be made in various tissues including liver, fat, and muscle. The mechanisms of insulin degradation in intact cells will be studied, in part using information obtained in the study of the purified enzyme, insulin protease. Thus the initial hypothesis, that is, that proteolytic processes are important in insulin degradation will be examined in intact cells.